The ability to transform cereal grains through recombinant techniques has important applications in the agricultural industry--for example, in achieving hardier crops, insect resistance, or higher yields, or for expression in plants of exogenous proteins of interest.
Methods for stably transforming barley (Hordeum vulgare ) have been demonstrated. These methods include, as example, the use of polyethylene glycol (PEG) or electroporation to facilitate DNA uptake by barley protoplasts, and particle bombardment of barley tissues, such as suspension cells, callus tissue and endosperm (Ritala, et al., Plant Cell Reports 12:435-440 (1993)). The challenge in these methods is to introduce foreign DNA into barley cells which are both totipotent and capable of stably integrating and expressing the foreign DNA.
Heretofore, stable transformation in barley has been achieved with cells (intact cells or protoplasts) obtained from early-stage embryos or surrounding scutellar tissue from early-growth seeds. Wan and Lemaux (Plant Physiol. 104:37-48 (1994)) reported successfully transforming barley plants using immature zygotic barley embryos as starting material. Chibbar et al. (WO 94/19930, published Sep. 15, 1994 ) disclose the regeneration of cereal plants, including barley, by introducing foreign into the scutellar tissue associated with immature zygotic embryos, typically obtained from early-development seeds 8-14 days post anthesis.
There is evidence that immature embryo cells are susceptible to stable transformation only during a short period in early embryogenesis (Wan, et al., supra). In order to prolong this period, and thus enhance the probability of successful transformation, it is standard practice currently to grow young barley plants under controlled temperature conditions, e.g., at 12-18.degree. C. This approach, however, is relatively expensive and time-consuming in that special incubators must be employed, and the time required for plant growth and early seed development is typically 3-4 months.
Thus, although methods for transforming barley have been available heretofore, there remains a need for a relatively inexpensive, short-time period method for producing stably transformed barley plants and cells.